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Title

Figure_3_ExperimentI

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Data collected in the laboratory, for the number of eggs spawned per female, over the 10 day study period of Experiment I. Each spawning group (each tank) contained 1 female and 2 males (either all inbred fish or all outbred as indicated in table). Low dose refers to previous exposure to 5 ug/L clotrimazole (nominal) as detailed in manuscript.

Data collected in the laboratory, for the number of eggs spawned per female, over the 10 day study period of Experiment II. Each spawning group (each tank) contained 1 female (either inbred or outbred, as indicated in table) and 2 males (one inbred and one outbred). Low dose refers to previous exposure to 5 ug/L clotrimazole (nominal) as detailed in manuscript.

Paternity analysis for Tank 1 (A21A). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 2 (A22A). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 3 (A23C). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 4 (A24C). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 5 (A25A). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 6 (A25B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 7 (A25C). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 8 (A26A). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 9 (A26B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 10 (A26C). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 11 (A27A). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 12 (A29B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 13 (A210B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 14 (B16B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 15 (B17A). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 16 (B17B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 17 (B18B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 18 (B19A). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Paternity analysis for Tank 19 (B19B). Study Day indicates the day on which each embryo analysed was spawned. The first 3 rows in the table are the parent fish and below that are the 20 selected offspring. The microsatellite loci used for analysis are listed along the top of the table and corresponding allelles are detailed below.

Data shows amount of plasma 11 ketotestosterone (ng/mL) measured in male fish, either inbred or outbred, or from the control or exposed treatment group, as indicated. The proportion of offspring sired by each male fish is also indicated (from spawning groups containing an outbred female only, as described in the associated manuscript).

AbstractInbreeding depression is expected to be more severe in stressful environments. However, the extent to which inbreeding affects the vulnerability of populations to environmental stressors, such as chemical exposure, remains unresolved. Here we report on the combined impacts of inbreeding and exposure to an endocrine disrupting chemical (the fungicide, clotrimazole) on zebrafish (Danio rerio). We show that whilst inbreeding can negatively affect reproductive traits, not all traits are affected equally. Inbreeding depression frequently only became apparent when fish were additionally stressed by chemical exposure. Embryo viability was significantly reduced in inbred exposed fish and there was a tendency for inbred males to sire fewer offspring when in direct competition with outbred individuals. Levels of plasma 11 keto-testosterone, a key male sex hormone, showed substantial inbreeding depression that was unaffected by addition of the fungicide. In contrast, there was no effect of inbreeding or clotrimazole exposure on egg production. Overall, our data provide evidence that stress may amplify the effects of inbreeding on key reproductive traits, particularly those associated with male fitness. This may have important implications when considering the consequences of exposure to chemical pollutants on the fitness of wild populations.